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(Slc26a7) by Thyroid Stimulating Hormone in Thyrocytes

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(Slc26a7) by Thyroid Stimulating Hormone in Thyrocytes 2021, 68 (6), 691-699 Original Regulation of solute carrier family 26 member 7 (Slc26a7) by thyroid stimulating hormone in thyrocytes Yuta Tanimura1), 2), Mitsuo Kiriya1), Akira Kawashima1), Hitomi Mori1), Yuqian Luo1), 3), Tetsuo Kondo2) and Koichi Suzuki1) 1) Department of Clinical Laboratory Science, Faculty of Medical Technology, Teikyo University, Itabashi, Tokyo 173-8605, Japan 2) Department of Pathology, Faculty of Medicine, University of Yamanashi, Chuo, Yamanashi 409-3898, Japan 3) Department of Laboratory Medicine, Nanjing Drum Tower Hospital and Jiangsu Key Laboratory for Molecular Medicine, Nanjing University Medical School, Nanjing 210008, China Abstract. Iodine transportation is an important step in thyroid hormone biosynthesis. Uptake of iodine into the thyroid follicle is mediated mainly by the basolateral sodium–iodide symporter (NIS or solute carrier family 5 member 5: SLC5A5), and iodine efflux across the apical membrane into the follicular lumen is mediated by pendrin (SLC26A4). In addition to these transporters, SLC26A7, which has recently been identified as a causative gene for congenital hypothyroidism, was found to encode a novel apical iodine transporter in the thyroid. Although SLC5A5 and SLC26A4 have been well-characterized, little is known about SLC26A7, including its regulation by TSH, the central hormone regulator of thyroid function. Using rat thyroid FRTL-5 cells, we showed that the mRNA levels of Slc26a7 and Slc26a4, two apical iodine transporters responsible for iodine efflux, were suppressed by TSH, whereas the mRNA level of Slc5a5 was induced. Forskolin and dibutyryl cAMP (dbcAMP) had the same effect as that of TSH on the mRNA levels of these transporters. TSH, forskolin and dbcAMP also had suppressive effects on SLC26A7 promoter activity, as assessed by luciferase reporter gene assays, and protein levels, as determined by Western blot analysis. TSH, forskolin and dbcAMP also induced strong localization of Slc26a7 to the cell membrane according to immunofluorescence staining and confocal laser scanning microscopy. Together, these results suggest that TSH suppresses the expression level of Slc26a7 but induces its accumulation at the cell membrane, where it functions as an iodine transporter. Key words: SLC26A7, SLC5A5, SLC26A4, TSH, Thyroid IODINE is a fundamental element for the biosynthesis circulating iodine into thyrocytes. SLC26A4 (or pendrin), of the thyroid hormones T3 and T4, and the prime func‐ another iodine transporter, was identified as a causative tion of the thyroid is to concentrate iodine and make it gene for Pendred syndrome in 1997, and the protein available for the biosynthesis of thyroid hormones. localizes on the apical membrane, opposite to SLC5A5 Iodine derived from dietary sources is transported into [2-4]. SLC26A7, encoding another member of the same the thyrocyte across the basal membrane and then transporter family as SLC26A4, was demonstrated to be a secreted across the apical membrane into the follicular novel causative gene for congenital hypothyroidism in lumen where it is organified to the tyrosine residues of 2018 [5-7]. Although SLC26A7 was first identified as a thyroglobulin, a large protein stored in the follicular chloride–bicarbonate anion exchanger in the kidney and colloid. Thus, the thyroid follicle serves as the minimal stomach [8], expression profiling of different human functional unit of hormone synthesis and secretion. tissues showed predominant mRNA expression of Sodium–iodide symporter (NIS or solute carrier SLC26A7 in the thyroid. Immunofluorescence staining of family 5 member 5: SLC5A5), the first iodine transporter thyroid tissue sections showed that SLC26A7 is local‐ cloned, in 1996 [1], is localized on the basolateral ized at the apical membrane, similar to pendrin [3, 5, 9]. membrane of thyrocytes and is responsible for taking up Bicameral culture system and radioiodine transport/ uptake studies suggested that SLC26A7 functions in the Submitted Aug. 8, 2020; Accepted Jan. 15, 2021 as EJ20-0502 efflux of iodine in culture medium [5], similar to pendrin Released online in J-STAGE as advance publication Feb. 14, 2021 [10]. SLC26A7 was thus considered a novel apical iodine Correspondence to: Koichi Suzuki, Ph.D., Department of Clinical Laboratory Science, Faculty of Medical Technology, Teikyo transporter responsible for iodine efflux in the thyroid. University, 2-11-1 Kaga, Itabashi, Tokyo 173-8605, Japan. TSH is the primary regulator of iodine uptake and E-mail: [email protected] stimulator of thyroid hormone production and secretion. ©The Japan Endocrine Society 692 Tanimura et al. TSH activates the Slc5a5 promoter to induce the expres‐ Slc26a7 reverse, 5'-CAAGCCACCTGTGTCTTTGC-3'; sion and membrane localization of the protein [9, 11-14]. NM_017008.4 Gapdh forward, 5'-ACAGCAACAGGGT It also induced the membrane abundance of SLC26A4 GGTGGAC-3'; Gapdh reverse, 5'-TTTGAGGGTGCAG and enhanced its function of iodine efflux into the folli‐ CGAACTT-3'. cular lumen, without affecting the total protein level [15, 16]. Although, the effects of TSH on SLC5A5 and Protein preparation and Western blot analysis SLC26A4 have been evaluated extensively, the role of Preparation of cellular proteins and Western blot TSH in the regulation of the newly identified SLC26A7 analysis were performed as described previously [19, 22, is not known. Here, we investigated the potential effect 25]. Briefly, cells were washed with ice-cold PBS and of TSH on Slc26a7 with respect to its expression and lysed in lysis buffer containing 20 mM Tris-HCl (pH subcellular localization in rat thyroid FRTL-5 cells. 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyro‐ Materials and Methods phosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4 and 1 μg/mL leupeptin. Cells were then scraped using a Cell culture and treatment disposable cell lifter (Corning, Corning, NY, USA) and Rat thyroid FRTL-5 cells were grown in Coon’s centrifugated. The supernatant was recovered, and pro‐ modified Ham’s F-12 medium supplemented with 5% tein concentrations were measured using the DC Protein bovine serum (Invitrogen, Waltham, MA, USA) and Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). a mixture of six hormones (1 mU/mL bovine TSH, Ten micrograms of each protein sample were separated 10 μg/mL insulin, 0.36 ng/mL hydrocortisone, 5 μg/mL by electrophoresis on NuPage 4–12% Bis-Tris gels transferrin, 10 ng/mL somatostatin and 2 ng/mL glycyl- (Invitrogen) and transferred to a nitrocellulose membrane L-histidyl-L-lysine acetate) as described previously using i-Blot gel transfer stacks (Invitrogen). The mem‐ [17-19]. After the cells attached to the bottom of plates brane was washed with 0.1% Tween 20 in PBS (PBST), (Greiner Bio-One, Kremsmünster, Austria), they were placed in blocking buffer (PBST containing 5% nonfat maintained in the same medium without TSH for 5 days, milk) for 1 h, and then incubated with mouse anti- followed by exposure to TSH, forskolin or dibutyryl Slc26a7 (1:1,000; Novus Biologicals, Centennial, CO, cAMP (dbcAMP; Sigma-Aldrich, St. Louis, MO, USA). USA) or mouse anti-β-actin (1:5,000; Cell Signaling Technology, Danvers, MA, USA) overnight. After Total RNA isolation and real-time PCR washing with PBST, the membrane was incubated with Total RNA was purified using the RNeasy Plus Mini horseradish peroxidase (HRP)-labelled horse anti-mouse Kit (Qiagen, Hilden, Germany), and cDNA was synthe‐ IgG (1:1,000; Cell Signaling Technology) for 1 h. The sized using the High-Capacity cDNA Reverse Transcrip‐ HRP was detected by chemiluminescence using the tion Kit (Applied Biosystems, Waltham, MA, USA) as ImmunoStar LD reagents (FUJIFILM Wako Pure Chem‐ described previously [20-22]. Real-time PCR was per‐ ical Corporation, Osaka, Japan) and analyzed by the C- formed using Fast SYBR Green Master Mix (Applied DiGit blot scanner (both from LI-COR, Lincoln, NE, Biosystems) according to the manufacturer’s instructions USA). and run on the Thermal Cycler Dice Real Time System III (Takara Bio, Tokyo, Japan). Briefly, 5 ng cDNA was Luciferase reporter gene assay mixed with 10 μL 2× Fast SYBR Green Master Mix and Among four transcript variants of SLC26A7, we used amplified by incubating for 30 sec at 95°C, followed by one that has the most upstream transcription start site 40 cycles of 5 sec at 95°C and 60 sec at 60°C, and 1 (NM_001282356.2). A series of sequences within the 5'- cycle of 15 sec at 95°C, 30 sec at 60°C and 15 sec at flanking region of the human SLC26A7 gene, starting at 95°C for dissociation. Real-time PCR was conducted in positions –2,207, –1,653, –1,140 and –443, relative to at least triplicate, and the relative mRNA expression the transcription start site, were amplified by PCR from levels were normalized to the corresponding Gapdh level human genomic DNA using the following forward and using the ∆∆Ct method as described [19, 23, 24]. The reverse primer pairs containing NheI or XhoI restriction sequences of the PCR primers are as follows: enzyme sites: –2,207 forward, 5'-CGTGCTAGCTGTGG NM_052983.2 Slc5a5 forward, 5'-CTACCGTGGGTGG CTAGGGATC-3'; –1,653 forward, 5'- CGTGCTAGCTA TATGAAGG-3'; Slc5a5 reverse, 5'-TGCCACCCACTAT GGGCTGTATAACAAAGTAT-3'; –1,140 forward, 5'- GAAAGTCC-3'; NM_019214.1 Slc26a4 forward, 5'- CGTGCTAGCATCTACTCAGTAGGGTGAG-3'; –443 CAAGTGGGTTCTTGCCTCCT-3'; Slc26a4 reverse, 5'- forward, 5'-CGTGCTAGCAGCACTGAAAAATCA TTGGTGGCGTAGACTTTCCC-3'; XM_008763531.2 A-3'; and reverse, 5'-GATCTCGAGACTTCCGTATTCT Slc26a7 forward, 5'-TGCTCCCCCAATGAACATCC-3'; TACCAGG-3'. The PCR products were introduced into Regulation of Slc26a7 in the thyroid 693 the pGL3-Basic luciferase reporter plasmid (Promega, PCR results clearly showed that TSH significantly sup‐ Madison, WI, USA) via the NheI and XhoI sites. FRTL-5 pressed Slc26a7 mRNA levels in a concentration- cells (2 × 105) maintained without TSH for 5 days in 6- dependent manner (Fig. 1A, left panel); on the other well plates (Greiner Bio-One) were transfected with luci‐ hand, TSH significantly induced Slc5a5 mRNA expres‐ ferase reporter plasmids using FuGENE HD transfection sion (Fig.
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